Subcellular Localization Of NGF In Glioma And Its Application In Diagnosis And Targeted Therapy

Objective:To observe the subcellular localization of nerve growth factor(NGF) in glioma and to explore its effect on the cell proliferation.NGF nuclear translocation is specific in glioma,this can be exploited to deliver the Auger electron emitter to the glioma cells for targeted radiotherapy.Methods:(1) The immunofluorescence staining and Western Blotting was used to localize the dynamic distributions of NGF and P-TrkA in different cell cycle phases of U251 cells.(2) MTT assay was used to observe the proliferation of U251 treated with NGF-β,anti-NGF and MG-132.(3) The immunofluorescence staining and Western Blotting was used to observe the nuclear translocation of NGF in glioma specimens excised by operation.To sum up the relation between translocation rate and malignant degree and prognosis.(4) NGF was radioiodinated with the Auger electron emitter ¹²⁵I by Bolton-Hunter method.Clone forming assay,Comet assay, Micronuclei forming assay and Flow cytometry were used to evaluate the anti-glioma activity of 125I-NGF.Results:(1) The results of immunofluorescence showed that NGF has a multitarget distribution in U251.After the mitotic cells were cultured for 6h,the NGF was distributed in peri-nuclei and the P-TrkA was located on the membrane,then the NGF and the P-TrkA were gradually translocated to nuclei;In the mitotic cells,the NGF was localized on the centrosomes which were immunofluorescence stained with antibody againstγ-tubulin,the P-TrkA was distributed on spindles which were immunofluorescence stained with antibody againstα-tubulin;In the cells arrested at G₁/S phase by treatment with hydroxyurea,the NGF and P-TrkA were localized in the nuclei; After treated with paclitaxel or colchicine,the NGF and P-TrkA were co-localized withγ-tubulin orα-tubulin.After cultured with the medium containing the serum of anti-NGF to neutralize the secretory NGF,the immunofluorescence staining of NGF and P-TrkA were weakened.The results of Westem Blotting showed that, in the medium cultured U251 cells,a specific NGF positive band of 36 kDa was detected,the bands of NGF and P-TrkA in nuclear extractions of the G₁/S phase were thicker than those in the cytoplasmic extractions of the cells.(2) MTT assay reveals that exogenous NGF can promote the proliferation of U251,while anti-NGF serum inhibits the proliferation of U251.(3) NGF nuclear translocation is common in glioma specimens,and this prognosticates the high malignant degree and bad clinical outcome.(4) ¹²⁵I-NGF is stable in vitro,it can be transported to nuclus.¹²⁵I-NGF is selectively radiotoxic to U251 cells at G₁/S phase,combination with hydroxyurea can enhance its action.Conclusion:1.This study suggested that NGF is overexpressed in U251 and its subcellular localization is highly phase dependent.2.Both endogenous and exogenous NGF can promote the proliferation of U251.3.Nuclear translocation of NGF is an index of glioma malignant degree and bad clinical outcome.Detection of 36kDa NGF in cerebrospinal fluid can be used for glioma early diagnosis and post-operation monitoring.4.¹²⁵I-NGF is a potential drug for targeted radiotherapy of glioma.